1. Field of the Invention
This invention relates to methods to forecast premature labor in a pregnant mammal and to prolong the term of pregnancy thereof to avoid preterm delivery.
2. Description of Related and Background Art
Inhibin and activin are members of a family of growth and differentiation factors. The prototype of this family is transforming growth factor-beta (TGF-.beta.). Derynck et al., Nature, 316: 701-705 (1985); Ying et al., Biochem. Biophys. Res. Commun., 135: 950-956 (1986). Other members of the TGF-.beta. family include the Mullerian inhibitory substance, the fly decapentaplegic gene complex, and the product of Xenopus Vg-1 mRNA.
Inhibin is a glycoprotein produced by diverse tissues, including the gonads, pituitary, brain, bone marrow, placenta, and adrenal gland. It was initially identified by its ability to inhibit the secretion of follicle stimulating hormone (FSH) by the pituitary. De Jong and Sharpe, Nature, 263: 71-72 (1976); Schwartz and Channing, Proc. Natl. Acad. Sci. USA, 74: 5721-5724 (1977). Such preferential regulation of the gonadotropin secretion has generated a great deal of interest and prompted many laboratories in the past fifty years to attempt to isolate and characterize this substance from extracts of testis, spermatozoa, rete testis fluid, seminal plasma, and ovarian follicular fluid using various bioassays. Rivier et al., Biochem. Biophys. Res. Commun., 133: 120 (1985); Ling et al., Proc. Natl. Acad. Sci. USA, 82: 7217 (1985); Fukuda et al., Mol. Cell Endocrinol., 44: 55 (1985). The structure of inhibin, characterized from several species, consists of two disulfide-linked subunits: an .alpha. and either a .beta..sub.A or a .beta..sub.B chain.
After the identification of inhibin, activin was shown to exist in follicular fluid as a naturally occurring substance. Activin was found to be capable of stimulating FSH release by rat anterior pituitary cells. Vale et al., Nature, 321: 776-779 (1986); Ling et al., Nature, 321: 779-782 (1986); DePaolo et al., Proc. Soc. Exp. Biol. Med., 198: 500-512 (1991); Ying, Endocrine Rev., 9: 267-293 (1988). Recombinant activin was also found to stimulate pituitary LH and FSH in the adult male macaque. McLachlan et al., Endocrinol., 125: 2787-2789 (1989). Activin consists of a homodimer or heterodimer of inhibin .beta. subunits, which may be .beta..sub.A or .beta..sub.B subunits. Vale et al., Recent Prog. Horm. Res., 44: 1-34 (1988). There is 95-100% amino acid conservation of .beta. subunits among human, porcine, bovine, and rat activins including the prepro region. The .beta..sub.A and .beta..sub.B subunits within a given species are about 64-70% homologous.
The inhibin heterodimers .alpha..beta..sub.A and .alpha..beta..sub.B ("Inhibin A" and "Inhibin B," respectively) and the activin .beta..sub.A and .beta..sub.B homodimers and activin .beta..sub.A .beta..sub.B heterodimer ("Activin A," "Activin B," and "Activin AB," respectively) have been identified in and purified from follicular fluid, and all these molecules have been cloned and their genes expressed. Mason et al., Biochem. Biophys. Res. Commun., 135: 957 (1986); U.S. Pat. No. 4,798,885 issued Jan. 17, 1989; Mason et al., Molecular Endocrinol., 3: 1352-1358 (1989); Schwall et al., Mol. Endocrinol., 2: 1237-1242 (1988); Nakamura et al., J. Biol. Chem., 267: 16385-16389 (1992). The complete sequence of the .beta..sub.B subunit is published in Serono Symposium Publications, entitled "Inhibin- Non-Steroidal Regulation of Follicle Stimulating Hormone Secretion," eds. Burger et al., abstract by Mason et al., vol. 42, pp. 77-88 (Raven Press, 1987), entitled "Human Inhibin and Activin: Structure and Recombinant Expression in Mammalian Cells." A method for purifying activin B is disclosed in U.S. Pat. No. 5,071,834. The recombinant activin molecule has been shown to increase serum levels of FSH in rats when delivered by subcutaneous injection. Schwall et al., Endocrinol., 125: 1420-1423 (1989); Rivier and Vale, Endocrinol., 129: 2463-2465 (1991).
Activin and inhibin regulate the growth and functions of a variety of cell types. They may be involved in diverse biological processes including erythropoiesis, bone formation, placental and gonadal steroidogenesis, neuronal survival, regulation of follicular matter, and embryologic mesodermal induction. In addition, activin has an effect on follicular granulosa cell differentiation (Sugino et al., Biochem. Biophys. Res. Commun., 153: 281-288 [1988]), spermatogonial proliferation (Mather et al., Endocrinol., 127: 3206-3214 [1990]), erythroid differentiation (EP 210,461 published Feb. 4, 1987 [where the protein is called BUF-3]; Eto et al., Biochem. Biophys. Res. Commun., 142: 1095-1103 [1987] and Murata et al., Proc. Natl. Acad. Sci. USA, 85: 2434-2438 [1988] [where the activin is called EDF]; Yu et al., Nature, 330: 765-767 [1987] [where the activin is called FRP]), stimulation of insulin secretion by pancreatic islets (Totsuka et al., Biochem. Biophys. Res. Commun., 156: 335-339 [1988]), enhancement of proliferation of fibroblast (Hedger et al., Mol. Cell Endocrinol., 61: 133-138 [1989]), stimulation of glucose production by hepatocytes (Mine et al., Endocrinology, 125: 586-591 [1989]), induction of a dose-dependent increase in inositol phosphates in rat parenchymal liver cells, an effect also seen with EGF (Mine et al., Biochem. Biophys. Res. Comm., 186: 205-210 [1992]), modulation of somatotroph functions (Billestrup et al., Mol. Endocrinol., 4: 356-362 [1990]), modulation of nerve cell differentiation (Schubert et al., Nature, 344: 868-870 [1990]; Hashimoto et al., Biochem. Biophys. Res. Comm., 173: 193-200 [1990]), and mesoderm induction. Smith et al., Nature, 345: 729-731 (1990); Mitrani et al., Cell, 63: 495-501 (1990).
The expression of inhibin subunits, each encoded by a separate gene, was demonstrated in several tissues in addition to ovary. Woodruff et al., Molec. Endocrinol., 1: 561-568 (1987). Inhibin .alpha., .beta..sub.A, and .beta..sub.B mRNAs were detected in testis, placental, spleen, pituitary, adrenal, bone marrow, and brain tissues. Meunier et al., Proc. Natl. Acad. Sci. USA, 85: 247-251 (1988). The expression of the inhibin subunit mRNAs varied by several-fold in a tissue-specific manner, suggesting different functions for these proteins depending on their pattern of association and their site of production. Activin mRNA (.beta..sub.A and .beta..sub.B subunits), bioactivity, and immunoactivity have been reported to be produced by testicular Leydig cells from immature rat and pig. Lee et al., Science, 243: 396-398 (1989); Lee et al., in Serono Symposium Publications, entitled "The Molecular and Cellular Endocrinology of the Testis" eds. Cooke and Sharpe, Vol. 50 (Raven Press: New York, 1988), p. 21-27.
Studies have shown that activin plays a role in embryo growth and differentiation. Mitrani et al., Cell, 63: 495-501 (1990); Roberts et al., Endocrinology, 128: 3122-3128 (1991); Smith et al., Nature, 345: 729-732 (1990); Thomsen et al., Cell, 63: 485-492 (1990); Van den Eijnden et al., Nature, 345: 732-734 (1990). Moreover, activin is synthesized by human trophoblast, maternal decidua, and fetal membranes (amnion and chorion) playing endocrine and paracrine roles in pregnancy. Petraglia et al., Science, 237: 187-189 (1987); Petraglia et al., J. Endocrinol. Metab., 71: 487-492 (1990); Petraglia et al., Am. J. Obstet. Gynecol., 165: 750-758; (1991); Rabinovici et al., J. Clin. Endocrinol. Metab., 75: 571-576 (1992); Petraglia et al., J. Clin. Endocrinol. Metab., 77: 542-548 (1993); Petraglia et al., J. Clin. Endocrinol. Metab., 74: 1184-1188 [1992]; Minami et al., Obstet. Gynecol., 80: 410-414 (1992); Petraglia et al., Proc. Natl. Acad. Sci. USA, 86: 5114-5117 [1989]; Manova et al., Mech. Dev., 36: 141-152 (1992); and Tanimoto et al., Biophys. Biochem. Res. Commun., 182: 773-778 (1992).
Activin beta A and B subunit mRNAs are expressed in human placental and decidual cells and mRNA levels increase throughout gestation, with highest values at term. Petraglia et al., J. Endocrinol. Metab., 71 supra; Petraglia et al., Am. J. Obstet. Gynecol. (1991), supra. Also, activin A is present in maternal circulation and levels increase throughout pregnancy, and activin A serum levels decline immediately following delivery, becoming undetectable within 6 hours postpartum. Petraglia, Endocr. J., 1: 323-327 (1993). Addition of human recombinant activin A increases the release of progesterone, GnRH, and hCG from cultured human placental cells [Petraglia et al., Proc. Natl. Acad. Sci. USA, 86: 5114-5117 (1989)] and of prostaglandin from cultured amnion cells [Petraglia et al., J. Clin. Endocrinol. Metab., 77, supra] and increases endothelin secretion by the placenta. Brown et al., Endocrinol., 129: 2355-2360 [1991].
In fact, an intense fluorescence signal for total immunoreactive inhibin/activin subunits and mRNAs have been identified in the epithelial layer of the amnion and chorion at term. Petraglia et al., J. Clin. Endo. Metab., 77, supra. See also Petraglia et al., J. Endocrinol. Invest., 16: 201-205 (1993) and Petraglia et al., Int. J. Gynecol. Obstet., 404 (abstract) (1991). For reviews on the subject, see Baird and Smith, Oxf. Rev. Reprod. Biol., 15: 191-232 (1993); Filicori, Curr. Opin. Obstet. Gynecol., 3: 309-315 (1991); deKretser et al., J. Endocrinol. Invest., 13: 611-624 (1990); deKretser et al., J. Endocrinol., 117 (suppl.) Abs. 14 (1988); Petraglia, in Hormone Gynecol. Endocrinol., ed. Genazzani et al. (Parthenon Publishers, Cornforth UK 1992), pp. 269-277.
It has been disclosed that there is an increased release of maternal activin A during parturition. Petraglia et al., Obstet. Gynecol. Survey, 48: 209-225 (1993); Norman et al., Fert. Steril., 59: 130-137 (1993). See also McLean et al., Obstet. Gynecol. Survey, 48: 209-225 [1993]. By newly developed specific two-site enzyme-linked immunosorbent assay (ELISA), activin A has been measured in maternal circulation, cord blood, and amniotic fluid. Petraglia et al., Obstet. Gynecol. Survey, supra. Circulating maternal serum activin A concentration increases throughout gestation, and cord blood activin A was measurable at term. Petraglia et al., Obstet. Gynecol. Survey, supra. According to a possible role around parturition, the expression of the .beta..sub.A subunit is maximum at term. Petraglia et al., J. Endocrinol. Metab., 71: 487-492 (1990). Activin A stimulates the release of prostaglandin E2 from amniotic cells, suggesting local paracrine/autocrine role. Petraglia et al., J. Clin. Endocrinol. Metab., 77, supra. Further, oxytocin (Sawchenko et al., Nature, 334: 615-617 [1988]) and ACTH (Plotsky et al., Endocrinol., 128: 2520-2525 [1991]) secretion is augmented in rats following activin administration and inhibited with .beta. subunit antibodies. Studies performed on cultured placental cells have shown no changes in activin A secretion following the incubation with relaxin or forskolin. Rabinovici et al., supra.
In granulosa cells, activin has been reported to inhibit (and TGF-.beta. to enhance) progesterone production. Ignotz and Massague, J. Biol. Chem.., 261: 4337 (1986). In primary cultures of granulosa cells, activin and inhibin as well as TGF-.beta. were found to affect hormone synthesis and secretion, each in a different fashion. Adashi and Resnick, Endocrinology, 119: 1879 (1986); Ying et al., Biochem. Biophys. Res. Commun., 136: 969 (1986); Hutchinson et al., Biochem. Biophys. Res. Commun., 146: 1405 (1987); Mondschein et al., Endocrinology, 123: 1970 (1988); Feng et al., J. Biol. Chem., 261: 14167 (1986). Inhibin .alpha. and .beta..sub.A subunit genes are expressed differentially in rat and mouse ovarian follicles during pregnancy. Penschow et al., J. Mol. Endocrinol., 4: 247-255 (1990). These molecules have both positive and negative effects on FSH-dependent granulosa cell function. Carson et al., J. Reprod. Fert., 85: 735-746 (1989). Also suggested is that individual members of the TGF-.beta./inhibin gene family regulate ovarian function, not only by direct action on follicle cells, but also indirectly by influencing the production rate of other members of that family. Zhiwen et al., Molecular and Cellular Endocrinology, 58: 161-166 (1988).
Activin and inhibin were reported to modulate growth of two gonadal cell lines, suggesting that these proteins may regulate proliferation as well as functions of gonadal cells. Gonzalez-Manchon and Vale, Endocrinology, 125: 1666-1672 (1989). It has now been shown that inhibin has a paracrine effect in stimulating ovarian follicular maturation [WO 91/10445] and that activin is useful for treating male infertility [U.S. Pat. No. 5,166,190], for treating polycystic ovarian disease when administered directly to the ovary [U.S. Pat. No. 5,102,868], for increasing the proportion of fertilized ova [U.S. Pat. No. 5,206,160], and for in vitro maturation of oocytes. PCT/US 94/02008.
Cell surface proteins that bind members of the TGF-.beta. superfamily have been identified. See, e.g., WO 92/20793 and the references cited below. The nature of ligand binding and signaling is complicated and it is this intricacy that allows the cell to respond in a variety of different ways. Discrete receptors for inhibin A, inhibin B, and activin B have not been identified. The divergence evolutionarily of inhibin versus the TGF-.beta. superfamily suggests that the receptor for this molecule may differ fundamentally from that of the activin binding moieties. Some of the identified orphan receptors may bind activin B.
The receptor subunits for the TGF-.beta. superfamily were first identified based on crosslinking studies of iodinated activin or TGF-.beta. to various cell types. A family of three binding moieties was identified using this methodology. The three membrane-derived binding moieties were named generically based on their molecular weight: Type I (55 kDa); Type II (70-85 kDa); and Type III (200-400 kDa).
The type III receptor was first identified as a beta-glycan present on the surface of most cells and having no signaling motif. See Wang et al., Cell, 67: 797-805 (1991). It is postulated that this cell surface binding protein binds TGF-.beta. on the cell surface and makes this molecule readily available to the type I:type II receptor complex.
The type II receptor was identified by expression cloning based on binding iodinated ligand. The first type II receptor for activin A was identified and cloned by Mathews and Vale, Cell, 65: 973-982 (1991). Attisano et al. reported that the activin type II receptor was highly homologous to a second family of activin type II receptor isoforms named activin type IIB.sub.1-4. Attisano et al., Cell, 68: 97-108 (1992). The unanticipated result of this research was that the type II receptors have a highly conserved serine-threonine kinase domain.
The type II receptors bind TGF-.beta. or activin A with a variety of affinities which may contribute to the narrow concentration dependence of activin effects on different cell types. For example, the fate of the Xenopus blastomere greatly differs when exposed to different concentrations (1.5-fold differences) of activin A. The expression of activin type IIB receptors (ActRIIB) has been described in Xenopus embryo, rat placenta, and human placenta. Jaffe, in Yen and Jaffe, eds., Reprod. Endocrinol. (Philadelphia: W B Saunders, 1991), pp. 758-769; Peng et al., Endocrinol., 133: 3046-3049 (1993). See also Roberts and Barth, Endocrinol., 134: 914-923 (1994) on expression of mRNA encoding the inhibin/activin system during mid- and late-gestation rat embryogenesis. The localization of ActRIIB mRNA receptors in the external syncytial layer of placental villi corresponds to the cells producing hCG and progesterone (Petraglia, Mol. and cell. Endocrin., 78: 109-112 [1991]), hormones whose secretion is modulated by activins. Petraglia et al., Proc. Natl. Acad. Sci. USA, 86: 5114-5117 (1989).
The type I receptor was identified by low-stringency PCR cloning based on the serine-threonine kinase domain. This strategy was the only method which could identify this component of the receptor complex because the type I receptor does not bind ligand in the absence of the type II subunit. Moreover, the signaling mechanism for activin/TGF-.beta. requires the association of ligand with both subunits. See Attisano et al., Cell, 75: 671-680 (1993); ten Dijke et al., Oncogene, 8: 2879-2887 (1993); Matsuzaki et al., J. Biol. Chem., 268: 12719-12723 (1993); Ebner et al., Science, 260: 1344-1348 (1993); and Wrana et al., Molec. Cell. Biol., 14: 944-950 (1994).
Receptors for other members of the TGF-.beta. superfamily have been identified based on PCR amplification of potential receptor members having similar serine-threonine kinase domains. A type I (daf-1) and type II (daf-4) receptor complex has been identified for BMP-2 and -4. Estevez et al., Nature, 365: 644-649 (1993). Lastly, four orphan human receptors have been identified which, based on their expression in mesenchymal cells adjacent to mullerian ducts during embryonic development, have been suggested to be MIS receptor isotypes. Matsuzaki et al., J. Biol. Chem., 268: 12719-12723 (1993); ten Dijke et al., Oncogene, 8: 2879-2887 (1993).
A new class of gonadal protein factors, named follistatin or FSH-suppressing protein (FSP), was isolated from side fractions derived from purifying porcine and bovine ovarian inhibins and activins. Ying, Endoc. Rev., 9: 267-293 (1988); Ling et al., "Isolation and characterization of gonadal polypeptides that regulate the secretion of follicle stimulating hormone," in Hodgen et al., eds., Non-Steroidal Gonadal Factors: Physiological Roles and Possibilities in Contraceptive Development, Jones Institute Press, Virginia, (1988), pp. 30-46. Follistatin was initially characterized by its ability to suppress FSH secretion from the pituitary. Thus, one biologic effect of follistatin is apparently similar to that of inhibin, but structurally the two proteins are quite different. Ueno et al., Proc. Natl. Acad. Sci. USA, 84: 8282-8286 (1987); Robertson et al., Biochem. Biophys. Res. Commun., 149: 744-749 (1987).
Follistatin is a glycosylated single-chain protein that is found in forms having molecular weights ranging from 31 to 39 kDa. All of these forms have similar amino acid compositions and identical amino-terminal amino acid sequences. The molecular cloning of cDNA with the gene of follistatin revealed two forms, a smaller molecular weight form and a larger form, which are generated by alternative splicing. The smaller form represents a carboxy-terminal truncated form of the larger precursor. For a review on follistatin and activin, see DePaolo et al., supra. Follistatin is now thought to be an inhibin/activin binding protein. See also Tuuri et al., J. Clin. Endo. Metab., 78: 1521-1524 (1994); Shi et al., Endocrinol., 134: 2431-2437 (1994); deKretser et al., Endocrinol., 134: 1231-1237 (1994); Petraglia et al., J. Clin. Endo. Metab., 78: 205-210 (1994); Mercado et al., Endocrinol., 132: 1774-1781 (1993); Torney et al., J. Endocrinol., 133: 111-120 (1992); Spencer et al., J. Clin. Endo. Metab., 71: 1678-1680 (1990); Kaiser et al., Endocrinol., 126: 2768-2770 (1990); Wongprasartsuk et al., J. Endocrinol., 141: 219-229 (1994).
Pregnant women with premature delivery have an increased risk of neonatal morbidity and death, showing a derangement of placental hormonal activity. Increased maternal concentration of relaxin, corticotropin-releasing factor, progesterone, or estrogen have been observed in association with preterm labor. McLean et al., supra; Norman et al., supra, Petersen et al., J. Obstet. Gynecol., 99: 292-295 [1992]; Warren et al., Am. J. Obstet. Gynecol., 166: 1198-1207 [1992]; Smith et al., Gynecol. Obstet. Invest., 18: 252 [1984]. Most preterm labors remain unexplained and the rate of preterm delivery remains unchanged at about 5%. McLean et al., Obstet. Gynecol. Survey, 48: 209-225 (1993). Despite the widespread use of tocolytic drugs there has not been any documented decrease in the rate of preterm birth in the last two years. This is largely due to the fact that there is still a very limited understanding of the physiology of parturition and the causes of preterm labor. Iams, J. Am. Med. Assoc., 262-265 (1989).
Since the development of a specific assay method, activin A is measurable in amniotic fluid and the highest concentrations have been found at labor. Petraglia et al., Endo. J., 1: 323-327 (1993). See also Baly et al., Endocrinol., 132: 2099-2108 (1993). Additionally, serum activin A levels increased significantly in pregnant women during vaginal or cesarean delivery after spontaneous labor. Petraglia et al., Obstet. and Gynecol., 84: 278-282 (1994). Bioactive and total immunoreactive inhibin has also been detected in amniotic fluid of pregnant women at term. Yohkichiya et al., Reprod. Fertil. Dev., 3: 671-678 (1991). All methods now available for measuring inhibin concentration recognize precursor molecules, mature forms of inhibin and free .alpha. subunit. Schneyer et al., J. Clin. Endocrinol. Metab., 70: 1208-1212 (1990). Amniotic fluid is a compartment where biochemical markers have been investigated. Prostaglandins, tumor necrosis factor, interleukin-1, and interleukin-6 amniotic concentrations are higher in women with preterm labor. Qu and Thomas, J. Clin. Endocrinol. Metab., 74: 1290-1295 (1992); Lopez et al., Br. J. Obstet. Gynecol., 94: 1156-1158 (1987); Romero et al., Am. J. Obstet. Gynecol., 166: 1576-1587 (1992); Romero et al., Am. J. Obstet. Gynecol., 160: 1117-1123 (1989); Romero et al., Am. J. Obstet. Gynecol., 169: 805-816 (1993). The introduction of a reliable prognostic biochemical marker would be of considerable value in women with preterm labor.
In addition, as noted above, there is clearly an unmet clinical need for preventing premature delivery of an infant.
Therefore, it is one object of the present invention to increase the length of a pregnancy so as to prolong delivery of an infant to avoid premature birth.
It is another object to provide a means for detecting whether premature labor is taking place by using activin A as a marker.
These and other objects will become apparent to one of ordinary skill in the art.